One of the remarkable features of chemical transmission between neurones is the ability of the neurone to selectively enhance the formation of transmitter in response to increased demand (i.e., release). In 5-hydroxythryptamine-containing neurones of the mammalian central nervous system, which are the subject of this investigation, one of the regulatory mechanisms for rapidly increasing transmitter synthesis is enhancement of the initial rate limiting step in 5-HT synthesis, namely the conversion of tryptophan to 5-hydroxytryptophan (5-HTP) by tryptophan hydroxylase. Recent in vitro experiments in this laboratory have shown that tryptophan hydroxylase becomes activated after deplorization of slices of rat brain stem in a potassium-enriched incubation medium. This activation is stable since it withstands the isolation procedures for the enzyme and freezing and thawing. The purpose of this proposal is to determine whether the enzyme also becomes activated and/or stimulated as a result of nerve impulse flow in vivo, and, if so, whether this activation reflects the ongoing level of activity in the 5-HT neurones. Experimental approaches to analyze this question will include electrical stimulation of 5-HT neurones in the midbrain raphe, electrolytic lesions of 5-HT perikarya in dorsal or median raphe to block nerve impulse flow, and the use of drugs in vivo and in vitro which have reproducibe and well characterized effects on firing of 5-HT neurones. Enzyme will be prepared from individual brain regions which receive projections from the midbrain raphe. Its activity (under subsaturating conditions) and kinetic properties will be determined with a very sensitive assay, utilizing high pressure liquid chromatography to isolate the 5-HTP formed, prior to fluorometric detection. If the enzyme does show altered kinetic properties to impulse flow or its absence, then it may be possible to use the state of activation of the enzyme as a bioassay for monitoring 5-HT neuronal activity in vivo, and in this way determine whether there is a selective involvement of particular groups of 5-HT neurones and their projections in specific physiological or pharmacological states.